Single cell transcriptomic profiling of a neuron-astrocyte assembloid tauopathy model

The use of iPSC derived brain organoid models to study neurodegenerative disease has been hampered by a lack of systems that accurately and expeditiously recapitulate pathogenesis in the context of neuron-glial interactions. Here we report development of a system, termed AstTau, which propagates toxic human tau oligomers in iPSC derived neuron-astrocyte assembloids. The AstTau system develops much of the neuronal and astrocytic pathology observed in tauopathies including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. Single cell transcriptomic profiling combined with immunochemistry characterizes a model system that can more closely recapitulate late-stage changes in adult neurodegeneration. The transcriptomic studies demonstrate striking changes in neuroinflammatory and heat shock protein (HSP) chaperone systems in the disease process. Treatment with the HSP90 inhibitor PU-H71 is used to address the putative dysfunctional HSP chaperone system and produces a strong reduction of pathology and neurodegeneration, highlighting the potential of AstTau as a rapid and reproducible tool for drug discovery.

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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. . Source data are provided with this paper. Source data are provided in the Source Data files as follows: Source data of differential gene expression results for Fig. 4c and Fig 7b, cell type markers for Suppl. Fig. 5c, and uncropped immunoblots for Fig. 3c,h, GProfiler2 FGSA results and gene set names for Fig. 5a, Fig. 7e, Fig. 8g, Suppl. Fig. 14h, Suppl. Fig. 15, and Suppl. Fig. 16, GProfiler2 FGSEA results in Cytoscape formatting for Fig. 5b and Suppl. Fig. 17a No statistical method was used to predetermine sample size. Sample size determination for immunolabeling quantification was based on prior studies with 3D assembloid modeling28,32,37. High-throughput scRNA-seq was selected to profile <1000 cells per replicate experimental condition providing a sufficiently large sample size for analysis.
No data were excluded from the immunolabeling analyses. Standard quality control filtration was performed in the analysis of the scRNA-seq data excluding cells with less than 200 and greater than 3000 detected genes or greater than 12% mitochondrial counts to remove low quality and multiplet cell reads.
All immunolabeling experiments were successfully repeated in at least 3 independent batches of asteroid cultures with at least 5 individual asteroids per quantification. scRNA-seq was successfully repeated in 4 independent batches of asteroid cultures.
Cultured 3D asteroids within a batch were blindly and randomly selected at timepoint collection for immunolabeling or scRNA-seq. Additionally, well plates were randomized for treatment to avoid marginal effects of cell growth on the plate. Further covariate control is not relevant to this study due to the highly controlled nature of the culture system.
Quantification of transcriptomics, granular intensity, MAP2 dendritic length, and immunoblot band intensity were blindly repeated by coauthors.